Sandro Rusconi

Metal binding 'finger' structures in the glucocorticoid receptor defined by site-directed mutagenesis.

The glucocorticoid receptor and the other members of the steroid receptor super‐family share a highly conserved, cysteine‐rich region which coincides with the DNA binding/transactivating domain. It has been postulated that this region is folded into two ‘zinc finger’ structures, similar to those originally reported for the transcription factor TFIIIA. The first potential finger domain contains four conserved cysteines and one conserved histidine, while the second contains five conserved cysteines. Using site‐directed mutagenesis, we have analysed the consequences of altering the proposed finger‐like structures. Our results show that most of the mutations affecting the conserved cysteines result in a total loss of glucocorticoid receptor function. In one important exception, however, a conserved cysteine (Cys500) is dispensable for glucocorticoid receptor activity and therefore cannot be involved in complexing a metal ion to form a finger structure. Moreover, the replacement of either Cys476 or Cys482 by His residues maintains partial in vivo activity of the glucocorticoid receptor, while their exchange for an alanine or serine residue, respectively, eliminates receptor function. These results support, at a genetic level, the involvement of cysteines of the glucocorticoid receptor DNA binding domain in metal ion complexation and define the candidate residues involved in such coordination.

Cloning of a Chironomus tentans cDNA encoding a protein (cEcRH) homologous to the Drosophila melanogaster ecdysteroid receptor (dEcR).

We have cloned a cDNA sequence coding for a Chironomus tentans steroid hormone receptor homologue which exhibits extensive amino acid sequence co-linearity with the ecdysteroid receptor of Drosophila melanogaster (dEcR; cell 67, 59-77). The DNA-binding domain has 95% and the hormone-binding domain 75% amino acid sequence identity with the cloned dEcR. The gene for this C. tentans protein is located on chromosome II, region 17C, as determined by in situ hybridization to polytene chromosomes of salivary glands. On Northern blots cDNA probes of the cloned gene hybridize to polyadenylated RNA of ca 4.2 kb. The expression of the cloned gene seems to be developmentally regulated and correlates to changes in ecdysteroid titer. Transfection of this C. tentans protein into D. melanogaster Schneiders line 2 cells leads to transcriptional interference with endogenous dEcR on an ecdysteroid-regulated promoter.

Interference and synergism of glucocorticoid receptor and octamer factors.

We have analysed the interplay of glucocorticoid receptor (GR) and the lymphocyte‐specific factor Oct‐2A with transient co‐transfection assays. Our data confirm our previously described observation that GR and the apparently unrelated factors belonging to the Octamer family can synergize when permitted to bind in cis. However, when GR binding sites are not present in the reporter genes, we observe that the action of the cloned factor Oct‐2A expressed in HeLa cells is strongly inhibited in the presence of active GR molecules. We can demonstrate that this GR‐mediated inhibition of Oct‐2A action is neither due to competitive binding to DNA target sites nor to a reduction of DNA binding competent Oct‐2A in the transfected cells. We observe that the phenomenon is not reciprocal, since co‐expression of Oct‐2A does not inhibit GR‐dependent transcription activation. Furthermore, we provide evidence that the observed GR‐Oct‐2A interference may be dependent on the type of cell line hosting the co‐transfected molecules. We consider it likely that the GR‐mediated inhibition is due to the exhaustion of some rate‐limiting co‐activators.

A single DNA-binding transcription factor is sufficient for activation from a distant enhancer and/or from a promoter position

Typical cell type‐specific or inducible mammalian genes are under the control of one or more remote enhancers which transmit their effect to the promoter region located at the initiation site of transcription. Both enhancers and promoters are composed of multiple binding sites for transcription factors. To study the requirements for promoter and enhancer function, we have used a reporter gene that is completely dependent on a single DNA‐binding transcription factor in vivo. This factor is a truncated, hormone‐independent form of the glucocorticoid receptor which interacts strongly with a palindromic binding site. After transfection into HeLa cells, transcription of a reporter gene with one, two or four copies of the binding site upstream of the TATA box is enhanced less than 10, at least 100 and greater than 1000‐fold respectively, in the presence of the receptor. Even when the TATA box is deleted, the four upstream binding sites confer receptor‐dependent transcription, though from scattered initiation sites. When four copies of the palindromic binding site are placed downstream of the transcription unit, they form a very strong receptor‐dependent enhancer. This enhancer can activate comparably well promoters containing binding sites for either glucocorticoid receptor, Sp1 factor, or octamer factor. Our data show that a single defined DNA‐binding factor can mediate both promoter and enhancer activity, and that it can co‐operate functionally both with itself and with seemingly unrelated transcription factors.

A novel expression assay to study transcriptional activators.

A novel assay to study transcriptional regulation in vivo designated trans-activation-dependent replication (TDR) assay is based on the modulation of a simian virus 40 (SV40)-derived replication system. A mixture of four plasmids (pPARA + pCIS + pTRANS + pREF) is co-transfected into vertebrate cells. After appropriate incubation, the replication of the pPARA plasmid (containing an SV40 origin of replication) is measured with a simple enzymatic test. We demonstrate that the level of replication is dependent on the differential trans-activation of the reporter pCIS (in which SV40 T-antigen is brought under control of the desired promoter) by the specific regulator protein encoded by the pTRANS plasmid. Three advantages make this assay a convenient tool for the systematic analysis of trans-activation in vivo: (1) remarkable sensitivity (higher than conventional assays); (2) rapid sample processing combined with a built-in standard (pREF-plasmid); (3) avoidance of expensive reagents such as freshly radiolabelled probes. We present the application of the TDR assay to the analysis of deletion mutants of the glucocorticoid receptor (GR) and other, GR-based chimeric trans-activators. The results demonstrate that the properties of protein domains are not always additive in a particular chimaera. Further application possibilities of the TDR assay are also discussed.

A transcriptional repressor obtained by alternative translation of a trinucleotide repeat.

Triplet nucleotide repeats are ubiquitous and rapidly evolving sequences in eucaryotic genomes. They are sporadically found in coding regions of transcription regulators where they become translated in different homopolymeric aminoacid (HPAA) stretches, depending on the local frame. Poly(CAG) yields three different HPAAs (poly Gln, Ser or Ala). Current sequence databases indicate a clear bias in the size and frequency of these HPAAs according to the rule: (Gln)n > (Ser)n >> (Ala)n. Aiming to understand the reasons of this bias, we changed the translational reading frame of the highly polymorphic CAG-repeat that normally encodes poly-Gln in the N-terminal portion of the rat glucocorticoid receptor (GR). The GR mutant in which the CAG repeat is translated to poly-Ala (called GR[Ala]) is incapable of transactivation, but maintains competence for hormone binding, nuclear translocation and specific DNA binding. We show that GR desactivation is obtained only when a very precise threshold length of the repeat is reached. GR[Ala] displays a strong negative dominance when tested for transcriptional activation in vivo and may become useful for selective competition of receptor dependent activities in tissue culture cells and transgenic animals. We discuss the implications of our findings for the understanding of the evolutionary behaviour of trinucleotide repeats in coding sequences.

Transcriptional repression by methylation: Cooperativity between a CpG cluster in the promoter and remote CpG-rich regions

Cytosine methylation of binding sites for transcription factors is a straightforward mechanism to prevent transcription, while data on an indirect mechanism, by methylation outside of the factor binding sites, are still scarce. We have studied the latter effect using a model promoter construct. For this, a 69 bp G + C rich DNA segment with a cluster of 14 CpG sites was inserted between upstream lexA sites and the TATA box. Transcription was measured in transient transfection assays with lexA‐VP16 as an activating factor. When the entire plasmid was methylated at all CpGs before transfection, transcription was blocked (to 3% residual activity), whereas transcription was only mildly inhibited (to 60%) by methylation of a control plasmid that lacked the 69 bp CpG cluster. However, the effect could not simply be attributed to methylation of the CpG cluster: neither a methylated CpG cluster in an otherwise methylation‐free reporter gene plasmid, nor the methylated plasmid with an unmethylated CpG cluster, inhibited transcription considerably (69% and 44% remaining activity, respectively). The data presented here suggest that a minimal length of methylated DNA in the promoter is required for repression, and imply that concomitant methylation of CpGs in the promoter region and in remote sequences can cooperatively block transcription, without the need to methylate any binding sites for transcription factors. We also note that the cooperation for a negative effect described here bears an analogy to transcriptional activation, where a promoter often cooperates with a remote enhancer.

Active, interactive, and inactive steroid receptor mutants.

In transient co-transfection assays, there is extensive cross-interaction between glucocorticoid receptor (GR) domains. For example, mutation of the conserved Ile residue at position 484 (rat GR map) to cysteine allows a net separation of transactivation and DNA binding. We also observed that the ligand binding domain plays a key role in cooperative transactivation. Furthermore, some carboxy-located mutations markedly alter the response of GR to agonists and antagonists. Finally, different reading frames of the CAG repeat that normally produces an amino-located poly-Gln repeat profoundly affect GR transactivation without altering DNA or ligand binding. This trans-dominant negative phenotype, seen when the CAG repeat yields a poly-Ala stretch, may turn out to be an excellent tool for functional analysis of GR in transgenic organisms.

Genetic and Biochemical Analysis of a Steroid Receptor

Extensive mutation studies of the glucocorticoid receptor (GR) and its target cis elements (GRE, 1) revealed a number of expected as well as some unexpected features of the molecular interactions underlying receptor mediated transcriptional control. We can show that constitutive GR fragments can work as promoter as well as “pure” enhancer factors. Furthermore, we propose an additional DNA target-discrimination role for the hormone binding domain of the GR.

Transactivation of the HIV promoter by Tat can be estimated by a bacterial blue-white color system

We describe an assay system which allows easy quantitation of transactivation of the human immunodeficiency virus (HIV) promoter by the viral Tat protein. We make use of the novel expression assay for the study of transcriptional activators [Rusconi et al., Gene 89 (1990) 211-221]. After transfection of a reference plasmid, a Tat expression plasmid, a plasmid in which the expression of simian virus 40 (SV40) large T antigen is driven by the HIV promoter and a replicator plasmid containing an SV40 ori into mammalian cells, low Mr DNA is shuttled back into Escherichia coli. Transactivation is quantitated by comparing the number of white colonies (due to the replicator plasmid) in presence and absence of Tat to the number of blue colonies (due to the reference plasmid). At high copy numbers of transfected reporter plasmid the system was saturated with respect to large T antigen and not accessible to transactivation by the viral Tat protein. Gradual decrease of the concentration of the HIV-promoter-containing plasmid resulted in continuous improvement of transactivation of this promoter. The demonstration of a 200-fold stimulation of the HIV-1 promoter indicates the sensitivity of the assay and its general applicability to analyse the interplay between a transacting factor and the responsive DNA/RNA sequences.